10/31/2017 1 Personalized Medicine : Genetic Testing and the Implications for future therapies Dr. Mohammad Omar Hussaini, MD Assistant Member, Moffitt Cancer Center Assistant Professor, University of South Florida Hematopathology and Molecular Pathology The body is made of many different types of cells. nitrogenous bases : - adenine c:r: thymine . guanine cytosine k ,, ....._ sugar phosphate backbone (a) base pair major groove minor groove 3' 5 ' 5' 3 ' (b) (c) The DNA code consists of 4 letters • A= adenine • T= thymine • C = cytosine • G= guanine
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10/31/2017
1
Personalized Medicine : Genetic
Testing and the Implications forfuture therapiesDr. Mohammad Omar Hussaini, MD
Assistant Member, Moffitt Cancer Center
Assistant Professor, University of South Florida
Hematopathology and Molecular Pathology
The body is made of many different types of
cells.
n itrogenous b ases:
- adenine
c:r: thym ine
...guanine
cytosinek,,....._
sugar
phosphate
b ackbone
(a)
base
pair
major
groove
m inor
groove
3' 5 '
5 ' 3 '
(b) (c)
The DNA code consists of 4 letters
• A= adenine
• T= thymine
• C = cytosine
• G= guanine
10/31/2017
2
Basics
• DNA contains 3 Gb (1 bp= 1 byte)
• 1% represents coding regions
• 25,000-30,000 genes
• Contain introns and exons
• Why sequence DNA?
• Central dogma of biology
Koboldt. Cell. 2013 Sep 26;155(1):27-38.
• DNA is the way that the cell stores information
• Provides the blueprint to make amino acids
• Amino acids come together to make proteins
• Proteins are important for the structure of cells, function of cells, and
regulation of cells
• If everything is working fine, the DNA contains all the information for
the cell to work normally
• However, if the something goes wrong in the DNA, then somethingcan go wrong in the cells and this results in disease
• Have to keep in mind that the whole story does not lie in the genome
only.
• Changes can be genetic (in genes) or
• Epigenetic
• Leave the gene sequence untouched
• Alters the gene activity (for example by methylation, acetylation, phosphorylation, ubiquitylation, chromatin modification and sumolyation)
• The changes can be inherited by daughter cells
• Can be influence by environment to turn on and off genes
Weinhold. Environ Health Perspect. 2006 Mar; 114(3):A160–A167.
A Modee of Clonal Ex1>1tnsion and Clonal Evoeution from N ounal Hematopoeis is toMyelod'ys,plasia a n d Myelo id Leuke'm ia
MDS
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• There can be a lot of mutations in a particular gene
• May not be cost effective, feasible, or reliable to detect all mutations
that may damage the protein
• We can measure the amount of protein or inability of protein tofunction
• Infer that there must be a mutation underlying that aberration
• Protein is derived from various tissue sample types
• For example:• Blood• Urine• Amniotic fluid
• Cerebrospinal fluid.
• Various methods can then be employed to analyze the protein function or amount:• high performance liquid chromatography (HPLC)• gas chromatography/mass spectrometry (GC/MS)• tandem mass spectrometry (MS/MS)
• Bioassays are functional and can employ flourometric, radioisotopic, or thin-layer chromatography methodologies.
GeneticAlliance; The New York-Mid-Atlantic Consortium
Jason M. Rizzo, and Michael J. Buck Cancer Prev Res2012;5:887-900
METHODOLOGY
Experimental Methodology: This test uses targeted next-generation sequencing to analyze coding regions of the most inclusive annotated RefSeq transcript for
each of the targeted genes. TruSeq Custom Amplicon assay was conducted for resequencing of enriched targeted regions. Sequenc ing of enriched libraries was
performed in multiplex on the Illumina MiSeq with paired-end, 150 base-pair configuration.
Informatics Methodology: There are four informatics tools used and relevant parameters used for each tool are detailed as follows:
Novoalign is an alignment tool. Samtools provides an input for Varscan. Varscan is a variant caller used to identify SNVs. Freebayes is a variant caller used to
identify insertions and deletions.
For single base-pair substitutions, an evaluation of this gene panel found a sensitivity of 99.32% for variant allele frequencies of 10-20% and a sensitivity of
100% for variant allele frequencies >20%. Specificity and positive predictive value were found to be 100% for substitutions with a variant allele frequency >10%.
Cutoff criteria were set such that a minimum variant allele frequency of 10% and a depth of 1000x were required to call single nucleotide variants.
For insertions and deletions, an evaluation of this gene panel identified 24 of 24 insertions and deletions with a variant allele frequency > 10%
Cutoff criteria were set such that a minimum variant allele frequency of 10% and a depth of 1000x were required to call insertions and deletions.
Note that it is possible that pathogenic variants may not be reported by one or more of the tools because of the parameters used. However, tool parameters were
optimized to maximize specificity and sensitivity.
Now that we know how to look; what do we
look for?Germline mutation testing
Dohner et al. Blood 2016 :blood-2016-08-733196
WHO classification
Classification*
Myeloid neoplasms with germ line predisposition without a preexisting disorder or organ dysfunction
AML with germ line CEBPA mutation
Myeloid neoplasms with germ line DDX41 mutation†
Myeloid neoplasms with germ line predisposition and preexisting platelet disorders
Myeloid neoplasms with germ line RUNX1 mutation†
Myeloid neoplasms with germ line ANKRD26 mutation†
Myeloid neoplasms with germ line ETV6 mutation†
Myeloid neoplasms with germ line predisposition and other organ dysfunction
Myeloid neoplasms with germ line GATA2 mutation
Myeloid neoplasms associated with bone marrow failure syndromes
Juvenile myelomonocytic leukemia associated with neurofibromatosis, Noonan syndrome, or Noonan syndrome-like disorders